Recent work suggests that heterotrimeric G proteins regulate and modulate important cell growth and differentiation pathways through interactions with cellular adapter proteins and kinases. The broad objective of this research proposal is to elucidate the molecular details governing protein-protein interactions in these relevant non-canonical signal transduction pathways. The proposed work is based on our recent discovery of a central role for a particular modular adaptor protein, p21-activated kinase interactive exchange factor (betaPIX) that links and regulates two signaling pathways important for tumor metastasis. BetaPIX appears to act as a scaffolding protein that sequesters kinases such as PAK and E3 ubiquitin ligases such as Cbl and AIP4. These signaling molecules regulate Rho GTPase family members and mediate a plethora of cellular processes including receptor internalization, regulation of cell size, apoptosis/survival, cellular morphology, and membrane trafficking. We hypothesize that betaPIX regulates G Protein Coupled Receptor (GPCR) - Receptor Tyrosine Kinase (RTK) cross talk in metastasizing breast cancer cells and propose that a novel CXCR4 signaling pathway exists in which G protein beta/gamma dimers are released to activate cellular signaling proteins bound to the betaPIX adaptor module. We will focus on two specific aims: (Aim 1) We will define the specificity and mechanism of the interaction between the betaPIX scaffold and its AIP4, PAK and Cbl binding partners and determine how formation and dissociation of these complexes are regulated. (Aim 2) We will determine how betaPIX scaffold complexes are regulated by G protein beta/gamma subunits generated from ligand-activated CXCR4 chemokine receptors. This research will be accomplished utilizing a multifaceted approach incorporating site-directed mutagenesis in combination with novel biophysical techniques including fluorescence spectroscopy, isothermal calorimetry (ITC) and X-ray crystallography in conjunction with in vivo FRET and BRET studies to identify key amino acids interactions that regulate the structural dynamics of these scaffolding complexes. Protein interactions with betaPIX comprise a key regulatory branch point for the cross talk between GPCR and RTK signal transduction pathways responsible for cancer cell metastasis. Understanding the molecular details controlling these interactions will be critical in devising clinical strategies to prevent tumor metastasis and better design of therapeutics to treat cancer. [unreadable] [unreadable] [unreadable]